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1.
Mol Ther ; 28(4): 1078-1091, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32053770

RESUMO

Caspase-8, a well-characterized initiator of apoptosis, has also been found to play non-apoptotic roles in cells. In this study, we reveal that caspase-8 can induce cell death in a special way, which does not depend on activation of caspases and mitochondrial initiation. Instead, we prove that caspase-8 can cause lysosomal deacidification and thus lysosomal membrane permeabilization. V-ATPase is a multi-subunit proton pump that acidifies the lumen of lysosome. Our results demonstrate that caspase-8 can bind to the V0 domain of lysosomal Vacuolar H+-ATPase (V-ATPase), but not the V1 domain, to block the assembly of functional V-ATPase and alkalinize lysosomes. We further demonstrate that the C-terminal of caspase-8 is mainly responsible for the interaction with V-ATPase and can suffice to inhibit survival of cancer cells. Interestingly, regardless of the protein level, it is the expression rate of caspase-8 that is the major cause of cell death. Taken together, we identify a previously unrevealed caspase-8-mediated cell death pathway different form typical apoptosis, which could render caspase-8 a particular physiological function and may be potentially applied in treatments for apoptosis-resistant cancers.

2.
J Exp Med ; 217(3)2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-31961917

RESUMO

Cancer cells often proliferate under hypoxia and reprogram their metabolism. However, how to find targets to effectively block the hypoxia-associated metabolic pathways remains unclear. Here, we developed a tool to conveniently calculate electrons dissipated in metabolic transformations. Based on the law of conservation of electrons in chemical reactions, we further built up an electron balance model for central carbon metabolism, and it can accurately outline metabolic plasticity under hypoxia. Our model specifies that glutamine metabolism reprogrammed for biosynthesis of lipid and/or proline actually acts as the alternative electron bin to enable electron transfer in proliferating cells under hypoxia. Inhibition of both proline biosynthesis and lipogenesis can synergistically suppress cancer cell growth under hypoxia and in vivo tumor onset. Therefore, our model helps to reveal combinations of potential targets to inhibit tumor growth by blocking hypoxia-rewired metabolism and provides a useful tool for future studies on cancer metabolism.

3.
Nat Commun ; 10(1): 201, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643150

RESUMO

Under hypoxia, most of glucose is converted to secretory lactate, which leads to the overuse of glutamine-carbon. However, under such a condition how glutamine nitrogen is disposed to avoid over-accumulating ammonia remains to be determined. Here we identify a metabolic flux of glutamine to secretory dihydroorotate, which is indispensable to glutamine-carbon metabolism under hypoxia. We found that glutamine nitrogen is necessary to nucleotide biosynthesis, but enriched in dihyroorotate and orotate rather than processing to its downstream uridine monophosphate under hypoxia. Dihyroorotate, not orotate, is then secreted out of cells. Furthermore, we found that the specific metabolic pathway occurs in vivo and is required for tumor growth. The identified metabolic pathway renders glutamine mainly to acetyl coenzyme A for lipogenesis, with the rest carbon and nitrogen being safely removed. Therefore, our results reveal how glutamine carbon and nitrogen are coordinatively metabolized under hypoxia, and provide a comprehensive understanding on glutamine metabolism.


Assuntos
Glutamina/metabolismo , Redes e Vias Metabólicas , Metaboloma , Neoplasias/metabolismo , Ácido Orótico/análogos & derivados , Acetilcoenzima A/metabolismo , Amônia/metabolismo , Amônia/toxicidade , Animais , Carbono/química , Carbono/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Glucose/metabolismo , Glutamina/química , Células HEK293 , Humanos , Ácido Láctico/metabolismo , Lipogênese , Metabolômica , Camundongos , Camundongos Nus , Neoplasias/sangue , Neoplasias/mortalidade , Neoplasias/patologia , Nitrogênio/química , Nitrogênio/metabolismo , Nucleotídeos/biossíntese , Ácido Orótico/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Cancer Biol Med ; 15(4): 389-399, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30891326

RESUMO

Objective: Pyruvate kinases M (PKM), including the PKM1 and PKM2 isoforms, are critical factors in glucose metabolism. PKM2 promotes aerobic glycolysis, a phenomenon known as " the Warburg effect". The purpose of this study was to identify the roles of PKM2 in regulating cellular metabolism. Methods: The CRISPR/Cas9 system was used to generate the PKM-knockout cell model to evaluate the role of PKM in cellular metabolism. Lactate levels were measured by the Vitros LAC slide method on an autoanalyzer and glucose levels were measured by the autoanalyzer AU5800. The metabolism of 13C6-glucose or 13C5-glutamine was evaluated by liquid chromatography/mass spectrometry analyses. The effects of PKM on tumor growth were detected in vivo in a tumor-bearing mouse model. Results: We found that both PKM1 and PKM2 enabled aerobic glycolysis, but PKM2 converted glucose to lactate much more efficiently than PKM1. As a result, PKM2 reduced glucose levels reserved for intracellular utilization, particularly for the production of citrate, and thus increased the α-ketoglutarate/citrate ratio to promote the generation of glutamine-derived acetyl-coenzyme A through the reductive pathway. Furthermore, reductive glutamine metabolism facilitated cell proliferation under hypoxia conditions, which supports in vivo tumor growth. In addition, PKM-deletion induced a reverse Warburg effect in tumor-associated stromal cells. Conclusions: PKM2 plays a critical role in promoting reductive glutamine metabolism and maintaining proton homeostasis. This study is helpful to increase the understanding of the physiological role of PKM2 in cancer cells.

5.
Hum Cell ; 28(1): 22-36, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25134797

RESUMO

This study aims to evaluate the effect of a human amniotic epithelial cell (HAEC)-rabbit corneal stroma tissue-engineered cornea on ocular reconstruction in three different animal models. HAECs were isolated from human placenta, seeded onto rabbit corneal stroma. HAECs-rabbit corneal stroma tissue engineering cornea transplantation was examined in three distinct rabbit models: transplantation of cornea constructed (1) with lamellar corneal HAECs and rabbit corneal stroma, (2) with central corneal HAECs and rabbit corneal stroma, or (3) with full-thickness corneal HAECs and rabbit corneal stroma. In the tissue engineering corneal transplantation groups in all three models, the mean number of days to corneal epithelial healing was significantly shorter than that in the control group and the mean number of days to corneal neovascularization was significantly greater than in the control group. In addition, in the tissue engineering corneal transplantation groups in the central lamellar cornea model and the full-thickness corneal transplantation model neovascularization, corneal turbidity, and epithelial fluorescence were significantly less than in the control groups. HAECs can be induced to differentiate into corneal epithelial cells, which may be suitable for the reconstruction of the corneal epithelium in cases of limbal stem cell deficiency.


Assuntos
Âmnio/citologia , Diferenciação Celular , Córnea/citologia , Doenças da Córnea/patologia , Doenças da Córnea/cirurgia , Transplante de Córnea/métodos , Células Epiteliais/citologia , Epitélio Anterior/citologia , Epitélio Anterior/cirurgia , Limbo da Córnea/patologia , Engenharia Tecidual/métodos , Animais , Substância Própria/citologia , Modelos Animais de Doenças , Humanos , Limbo da Córnea/citologia , Coelhos , Transplante de Células-Tronco , Células-Tronco/patologia
6.
Anal Chim Acta ; 697(1-2): 83-9, 2011 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-21641422

RESUMO

The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 ± 4.62 nm to 129.70 ± 43.72 nm) and the expression of CD44 decreased (99.79 ± 0.16% to 75.14 ± 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 µM curcumin-treated) and 50-120 pN (20 µM curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.


Assuntos
Anticorpos/imunologia , Reações Antígeno-Anticorpo/efeitos dos fármacos , Antineoplásicos/farmacologia , Curcumina/farmacologia , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Nanoestruturas , Apoptose/efeitos dos fármacos , Fenômenos Biomecânicos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Receptores de Hialuronatos/química , Microscopia de Força Atômica , Transporte Proteico/efeitos dos fármacos
7.
Eur J Pharmacol ; 650(1): 41-7, 2011 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-20883687

RESUMO

Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24h. The 50% inhibiting concentration (IC50) was 17.5±3.2µM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca(2+) concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Curcumina/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos
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